WO2001072742A1

WO2001072742A1 - Bisamidino compounds as nhe-3 inhibitors

Info

Publication number: WO2001072742A1
Application number: PCT/EP2001/003474
Authority: WO
Inventors: Rolf Gericke; Norbert Beier; Claudia Wilm; Peter Raddatz
Original assignee: Merck Patent Gmbh
Priority date: 2000-03-28
Filing date: 2001-03-27
Publication date: 2001-10-04
Also published as: AR027734A1; DE10015248A1; AU6381701A

Abstract

The invention relates to compounds of formula (I), wherein X and Y have the meanings given in claim number 1. Said compounds are inhibitors of the sodium/proton exchanger subtype 3 (NHE-3).

Description

Bisamidino compounds as NHE-3 inhibitors

The invention relates to the use of compounds of the formula

in what mean

n: an integer value between 1 and 6

and their salts and solvates as NHE inhibitors.

Sodium / proton exchangers or antiporters are transport proteins that are arranged in the plasma membrane and exchange extracellular Na ⁺ for intracellular H ⁺ . These proteins are found in all animals. The Na ⁺ / H ⁺ exchangers form a family with at least six different isoforms (NHE-1 to NHE-6), all of which have now been cloned. While the NHE-1 subtype is ubiquitously distributed throughout the body in all tissues, the remaining NHE subtypes become selective in specific organs such as in the kidney or in the lumen wall and Contraluminal wall of the small intestine expressed. This distribution reflects the specific functions that the different isoforms serve, namely on the one hand the regulation of the intracellular pH and the cell volume by the subtype NHE-1 and on the other hand the Na ⁺ -

Absorption and reuptake in the intestine and kidney through the isoforms

NHE-2 or NHE-3. The NHE-4 isoform is mainly found in the stomach. The expression of NHE-5 is limited to brain and neuronal tissue. NHE-6 represents the isoform that forms the sodium / proton exchanger in the mitochondria. The NHE-3 isoform is particularly expressed in the apical membrane of the proximal kidney tubules. An NHE-3 inhibitor therefore exercises a kidney protection effect.

The therapeutic use of a selective inhibitor for NHE-3 isoforms is versatile. NHE-3 inhibitors inhibit or reduce tissue damage and cell necrosis following pathophysiological hypoxic and ischemic events that lead to activation of NHE activity, such as during renal ischemia or during kidney transplant removal, transport, and reperfusion during kidney transplantation is.

Inhibitors of the sodium / proton exchanger subtype 3 are described, for example, in EP 0 825 178.

The object of the invention is to provide new inhibitors of the sodium / proton exchanger.

This object is achieved by using compounds of the formula I shown above as NHE inhibitors. The compounds show a high NHE-3 activity combined with a pronounced selectivity towards the NHE-1 and NHE-2 isoforms. Bisamidino compounds of the formula I are known in principle. Representatives of these classes of substances are described, for example, by O. Dann, H. Char and H. Griesmayer, Liebigs Ann. Chem. 1982, 1836 to 1869, O. Then, H. Char, P. Fleischmann, H. Fricke, Liebigs Ann. Chem. 1986, 438 to 455 and O. Then, G. Volz, E. Demant, W. Pfeifer, G. Bergen, H. Fick, E. Walkenhorst, Liebigs Ann. Chem. 1973, 1112 to 1140. The authors also examined the trypanocidal and antileukaemic properties of these substances.

The compounds of formula I lower blood pressure and are suitable as active pharmaceutical ingredients for the treatment of hypertension. They are also suitable as diuretics.

The compounds of formula I, alone or in combination with NHE inhibitors of other subtype specificity, have anti-ischemic effects and can be used in thromboses, atherosclerosis, vascular spasms, for protecting organs, e.g. Kidney and liver, before and during operations, as well as with chronic or acute kidney failure.

They can also be used to treat stroke, himedema, ischemia of the nervous system, various forms of shock and to improve respiratory drive in, for example, the following conditions: central sleep apnea, sudden child death, postoperative hypoxia and other breathing disorders.

Combination with a carbonic anhydrase inhibitor can further improve breathing.

The compounds of the formula I have an inhibitory effect on the proliferation of cells, for example fibroblast cell proliferation and the proliferation of smooth vascular muscle cells and can therefore be used for treatment used in diseases in which cell proliferation is a primary or secondary cause.

The compounds of formula I can be used against diabetic late complications, cancer, fibrotic diseases, endothelial dysfunction, organ hypertrophy and hyperplasia, especially in prostate hyperplasia or prostate hypertrophy.

They are also suitable as diagnostics for the determination and differentiation of certain forms of hypertension, atherosclerosis, diabetes and proliferative diseases.

Since the compounds of the formula I also have an advantageous effect on the level of the serum lipoproteins, they can be used alone or in combination with other medicaments for the treatment of an elevated blood fat level.

The invention relates to the use of compounds of

Formula I according to claim 1 and its physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of thromboses, ischemic conditions of the heart, peripheral and central nervous system and stroke, ischemic conditions of peripheral organs and limbs and for the treatment of shock conditions.

The invention further relates to the use of compounds of the formula I and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of diseases in which cell proliferation is a primary or secondary cause, for the treatment or prophylaxis of disorders of the fat metabolism or disturbed breath drive. The invention further relates to the use of compounds of the formula I and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of ischemic kidney, ischemic bowel disease or for the prophylaxis of acute or chronic kidney disease.

Methods for the identification of substances that inhibit the sodium / proton exchanger subtype 3 are e.g. in US 5,871,919.

Hydrates are e.g. the hemi, mono- or dihydrates, among solvates e.g. Alcohol addition compounds such as with methanol or ethanol.

The following compounds have proven to be particularly advantageous in their action as NHE-3 inhibitors:

a) 2,2-tetramethylene di-1-benzofuran-5-carboxamidine (1); b) 2,2-hexamethylene di-1-benzofuran ~ 5-carboxamidine (2); c) 2- [2- (6-Amidinoindol-2-yl) ethyl] -1-benzofuran-5-carboxamidine (3); d) trans-1,2-bis (5-amidino-2-benzofuranyl) ethylene (4);

and their salts and solvates.

The compounds of the formula I and also the starting materials for their preparation are otherwise prepared by methods known per se, as described in the literature (for example in the standard works such as Houben-Weyl, methods of organic chemistry, Georg-Thieme-Verlag, Stuttgart) are described, namely under reaction conditions which are known and suitable for the reactions mentioned. It is also possible to use variants which are known per se and are not mentioned here in detail

Make use. If desired, the starting materials can also be formed in situ, so that they are not isolated from the reaction mixture, but instead are immediately reacted further to give the compounds of the formula I.

The compounds of the formula I are preferred according to the method described by 0. Dann et al. prepared in the methods described above.

The amidino group is preferred by converting a cyano group by reaction with e.g. Hydroxylamine and subsequent reduction of the N-hydroxyamidine with hydrogen in the presence of a catalyst such as e.g. Obtain Pd / C or Raney nickel. To produce the amidine group, ammonia can also be added to a corresponding nitrile. The addition is preferably carried out in several stages, in a manner known per se

a) converts the nitrile with H ₂ S into a thioamide, which is converted with an alkylating agent, eg CH ₃ I, into the corresponding S-alkyl imidothioester, which in turn reacts with NH ₃ to form the amidine, b) the nitrile with an alcohol, for example ethanol in the presence of HCl, is converted into the corresponding imidoester and treated with ammonia, or c) the nitrile is reacted with lithium bis (trimethylsilyl) amide and the product is then hydrolyzed.

A base of the formula I can be converted into the associated acid addition salt using an acid, for example by reacting equivalent amounts of the base and the acid in an inert solvent such as ethanol and subsequent evaporation. In particular, acids that provide physiologically acceptable salts are suitable for this implementation. So inorganic acids can be used, e.g.

Sulfuric acid, nitric acid, hydrogen halide acids such as hydrochloric acid hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, furthermore organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or polybasic carboxylic, sulfonic or sulfuric acids, for example formic acid, acetic acid, propionic acid, pivalic acid, diethyl acetic acid .

Malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethanesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic, naphthalene-mono- and -disulfonic acids, lauryl sulfuric acid. Salts with physiologically unacceptable acids, e.g. Picrates can be used for the isolation and / or purification of the compounds of the formula I.

On the other hand, compounds of formula I with bases (e.g. sodium or potassium hydroxide or carbonate) can be converted into the corresponding metal, in particular alkali metal or alkaline earth metal, or into the corresponding ammonium salts. Also physiologically harmless organic bases, e.g. Ethanolamine can be used.

The invention furthermore relates to the use of the compounds of the formula I as NHE-3 inhibitors and / or their physiologically acceptable salts for the production of pharmaceutical preparations, in particular in a non-chemical way. Here, they can be brought into a suitable dosage form together with at least one solid, liquid and / or semi-liquid carrier or auxiliary and optionally in combination with one or more other active substances.

The invention further relates to pharmaceutical preparations containing at least one NHE-3 inhibitor of the formula I and / or one of its physiologically acceptable salts and solvates.

These preparations can be used as medicinal products in human or veterinary medicine. Suitable carriers are organic or inorganic substances which are suitable for enteral (for example oral), are suitable for parenteral or topical application and do not react with the new compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, petroleum jelly. Tablets, pills, coated tablets,

Capsules, powders, granules, syrups, juices or drops, for rectal use suppositories, for parenteral use solutions, preferably oily or aqueous solutions, further suspensions, emulsions or implants, for topical use ointments, creams or powder, or transdermally in patches.

The compounds can also be lyophilized and the lyophilizates obtained e.g. can be used for the production of injectables. The specified preparations can be sterilized and / or auxiliary substances such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, coloring, taste and / or one or more contain other active ingredients, e.g. one or more vitamins.

Suitable pharmaceutical preparations for administration in the form of aerosols or sprays are e.g. Solutions, suspensions or emulsions of the active ingredient of formula I in a pharmaceutically acceptable solvent.

The compounds of formula I and their physiologically acceptable

Salts and solvates can be used to treat and / or prevent the diseases or conditions described above.

The substances according to the invention are generally preferably administered in doses between about 0.1 and 100 mg, in particular between 1 and 10 mg, per dosage unit. The daily dosage is preferably between about 0.001 and 10 mg / kg body weight. However, the specific dose for each patient depends on a wide variety of factors, for example on the effectiveness of the special compound used, on the age, body weight, general health, gender, on the diet, on the administration time. point and route, on the rate of excretion, drug combination and severity of the respective disease to which the therapy applies. Oral application is preferred.

The invention is explained in more detail by means of examples.

Synthesis of the active ingredients

According to O., 2,2'-tetramethylenedi-1-benzofuran-5-carboxamidine (1) was

Then, H. Char, H. Griesmayer, Liebigs Ann. Chem. 1982, 1836 to 1869.

2,2'-Hexamethylenedi-1-benzofuran-5-carboxamidine (2) was prepared analogously to this synthesis.

2- [2- (6-Amidinoindol-2-yl) ethyl] -1-benzofuran-5-carboxamidine (3) was prepared according to the instructions in O. Dann, H. Char, P. Fleischmann, H. Fricke, Liebigs Ann , Chem. 1986, 438 to 455.

Trans-1,2-bis (5-amidino-2-benzofuranyl) ethylene (4) was prepared according to the instructions of O. Dann, G. Volz, E. Demant, W. Pfeifer, G. Bergen, H. Fick, E. Walkenhorst Liebigs Ann. Chem. 1973, 1112 to 1140.

The dihydrochlorides were used for the pharmacological tests.

Pharmacological tests

The methodology used to characterize the compounds of the formula I as NHE-3 inhibitors is shown below. The compounds of the formula I were characterized with regard to their selectivity towards the isoforms NHE-1 to NHE-3. The three isoforms were stably expressed in mouse fibroblast cell lines. The inhibitory activity of the compounds was assessed by determining the ElPA-sensitive ²² Na ⁺ uptake into the cells after intracellular acidosis.

material and methods

LAP1 cell lines that express the different NHE isoforms

The LAP1 cells expressing the NHE-1, -2 and -3 isoforms (a mouse fibroblast cell line) were obtained from Prof. J. Pouyssegur (Nice, France). The transfections were carried out according to the method of Franchi et al. (1986). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% inactivated fetal calf serum (FCS). For the selection of the NHE-expressing cells, the so-called "acid killing method" by Sardet et al. (1989) used. The cells were first incubated for 30 minutes in a NH ₄ CI-containing bicarbonate and sodium-free buffer. The extracellular NH CI was then removed by washing with a buffer free of bicarbonate, NH ₄ CI and sodium. The cells were then incubated in a bicarbonate-free NaCl-containing buffer. Only those cells that functionally express NHE were able to survive in the intracellular acidification to which they were exposed.

Characterization of NHE inhibitors in terms of their isoform selectivity

With the above mouse fibroblast cell lines expressing the isoforms NHE-1, NHE-2 and NHE-3, compounds according to the method described by Counillon et al. (1993) and Scholz et al. (1995) described procedure for selectivity towards the isoforms. The cells were acidified intracellularly using the NH ₄ CI prepulse method and then by incubation in a bicarbonate-free ²² Na ⁺ -containing buffer. NHE was activated due to the intracellular acidification and sodium was taken up into the cells. The effect of the test compound was expressed as an inhibition of EIPA (ethyl isopropylamiloride) sensitive ²² Na ⁺ uptake.

The cells expressing NHE-1, NHE-2 and NHE-3 were in one

Density of 5-7.5 x 10 ⁴ cells / wells in microtiter plates with 24 wells sown and grown to confluence for 24 to 48 hours. The medium was aspirated and the cells were incubated for 60 minutes at 37 ° C. in the NH ₄ CI buffer (50 mM NH ₄ CI, 70 mM choline chloride, 15 mM MOPS, pH 7.0). The buffer was then removed and the cells were rapidly overlaid twice with the choline chloride washing buffer (120 mM choline chloride, 15 mM PIPES / Tris, 0.1 mM ouabain, 1 mM MgCl ₂ , 2 mM CaCl ₂ , pH 7.4); the cells were incubated in this buffer for 6 minutes. After the incubation period had elapsed, the incubation buffer was aspirated. To remove extracellular radioactivity, the cells were quickly washed four times with ice-cold phosphate-buffered saline (PBS). The cells were then solubilized by adding 0.3 ml of 0.1 N NaOH per well. The cell fragment-containing solutions were transferred to scintillation tubes. Each well was washed twice more with 0.3 ml of 0.1 N NaOH and the washing solutions were also added to the corresponding scintillation tubes. Scintillation cocktail was added to the tubes containing the cell lysate and the radioactivity absorbed into the cells was determined by determining the β radiation.

Literature:

Counillon et al. (1993) Mol. Pharmacol. 44: 1041-1045 Franchi et al. (1986) Proc. Natl. Acad. Be. USA 83: 9388-9392 Morgan and Canessa (1990) J. Membrane Biol. 118, 193-214

Sardet et al. (1989) Cell 56: 271-280 Scholz et al. (1995) Cardiovasc. Res. 29: 260-268. test results

IC50 (NHE-1) = 2.2μM IC50 (NHE-3) = 1.7μM

IC50 (NHE-1) = 2.7μM IC50 (NHE-3) = 1.9μM

IC50 (NHE-1) = 4.6M IC50 (NHE-2) = 47.6μM IC50 (NHE-3) = 0.98μM

IC50 (NHE-1) = 0.69 µM IC50 (NHE-3) = 1.5 µM

0

Example A: Injection glasses

A solution of 100 g of an NHE-3 inhibitor of the formula I and 5 g of disodium hydrogenphosphate is adjusted to pH 6.5 in 3 l of double-distilled water with 2N hydrochloric acid, sterile filtered, filled into injection glasses and lyophilized under sterile conditions and sealed sterile. Each injection jar contains 5 mg of active ingredient.

⁰ Example B: suppositories

A mixture of 20 g of an NHE-3 inhibitor of the formula I is melted with 100 g of soy lecithin and 1400 g of cocoa butter, poured into molds and allowed to cool. Each suppository contains 20 mg of active ingredient. 5

Example C: solution

A solution of 1 g is prepared of an NHE-3 inhibitor of the formula I, 9.38 g NaH2Pθ4 ^• 2 H2O, 28.48 g of Na2HP04 ^• 12 H2O and 0.1 g of benzalkonium ^υ koniumchlorid in 940 ml of double-distilled water. It is adjusted to pH 6.8, made up to 1 I and sterilized by irradiation. This solution can be used in the form of eye drops.

Example D: Ointment 5 500 mg of an NHE-3 inhibitor of the formula I are mixed with 99.5 g of petroleum jelly under aseptic conditions.

Example E: tablets

A mixture of 1 kg of an NHE-3 inhibitor of the formula I, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is compressed into tablets in a conventional manner such that each tablet 10 mg contains active ingredient.

Example F: coated tablets

Analogously to Example E, tablets are pressed, which are then coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and colorant.

Example G: capsules

2 kg of an NHE-3 inhibitor of the formula I are filled into hard gelatin capsules in a conventional manner, so that each capsule contains 20 mg of the active ingredient.

Example H: ampoules

A solution of 1 kg of NHE-3 inhibitor of the formula I in 60 l of double-distilled water is sterile filtered, filled into ampoules, lyophilized under sterile conditions and sealed sterile. Each ampoule contains 10 mg of active ingredient.

Claims

claims

1. Compounds of the formula

in which mean

n: an integer value between 1 and 6;

and their salts and solvates as NHE-3 inhibitors.

Compounds according to claim 1: a) 2,2'-tetramethylene di-1-benzofuran-5-carboxamidine (1); b) 2,2'-hexamethylenedi-1-benzofuran-5-carboxamidine (2); c) 2- [2- (6-Amidinoindol-2-yl) ethyl] -1-benzofuran-5-carboxamidine

(3); d) trans-1,

2-bis (5-amidino-2-benzofuranyl) ethylene (4) and their salts and solvates as NHE-3 inhibitors.

3. Use of compounds of formula I according to claim 1 and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of thromboses, ischemic conditions of the heart, the peripheral and central

Nervous system and stroke, ischemic conditions of peripheral organs and limbs and for the treatment of shock conditions.

4. Use of compounds of formula I according to claim 1 and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for use in surgical operations and organ transplants and for the preservation and storage of transplants for surgical measures.

Use of compounds of formula I according to claim 1 and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of diseases in which cell proliferation is a primary or secondary cause, for the treatment or prophylaxis of disorders of the fat metabolism or impaired respiratory drive.

6. Use of compounds of formula I according to claim 1 and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of ischemic kidney, ischemic bowel disease or for the prophylaxis of acute or chronic kidney disease.

7. Pharmaceutical preparation, characterized by a content of at least one NHE-3 inhibitor according to claim 1 and / or one of its physiologically acceptable salts and / or solvates.